Abstract
The benzenearsonate, Roxarsone, has been used since 1944 as an antimicrobial, growth-promoting poultry feed additive. USGS and EPA report that Roxarsone (4-hydroxy-3-nitrobenzenearsonate) and metabolites, including AHBA (3-amino-4-hydroxybenzenearsonate), contaminate waterways at greater than 1100 tons annually. To assess human impact of these organic arsenic water contaminants, it was important to study their potential absorption. The human adenocarcinoma cell line, Caco-2, is a model for intestinal absorption. We found proliferative effects on Caco-2 cells at micromolar levels of these compounds, as monitored by [3H]-thymidine incorporation into DNA. Flow cytometry cell cycle analysis confirmed accumulation in S phase from 21% (control) to 36% (24 hour exposure to 10 μM AHBA). Confluent Caco-2 cells grown on collagen-coated Transwell plates were dosed on the apical side. After exposure, media from apical and basolateral sides were collected separately. Following removal of FBS by 30K centrifugal filtration, the benzenearsonates in the collected media were analyzed by HPLC. Analyses were at wavelengths in the ultraviolet/visible range where the absorbance values were linear with respect to concentration. Concentrations were calculated by comparison with analytically-prepared commercial standards. Results from cells dosed at 10 μM for 24 hours with AHBA, Roxarsone, or Acetarsone indicated 6% - 29% permeation occurring from apical to basolateral side, modeling absorption across intestinal epithelium to the circulatory system. Benzenearsonate feed additives are frequently applied in combination with antibiotics, raising additional health concerns. We conclude that micromolar levels of these benzenearsonates are adequate to stimulate Caco-2 cell proliferation.
Highlights
The number of concentrated animal feeding operations (CAFOs) has increased in the US since the late 1980s
There are several literature reports of Roxarsone and two metabolic products, the nitro-reduced AHBA arising from microbial sources [13], and the N-acetylated Acetarsone from animal [12] and human sources [23]
The proliferative effects of Roxarsone, AHBA, and Acetarsone on synchronized Caco-2 cells were examined using two independent methods, cell proliferation monitored by [3H]-thymidine uptake and cell cycle analysis using flow cytometry. [3H]-thymidine uptake into DNA as described in MATERIALS AND METHODS was used as a measure of DNA synthesis primarily occurring during the S phase of the cell cycle
Summary
The number of concentrated animal feeding operations (CAFOs) has increased in the US since the late 1980s. From the 1940s, benzenearsonates have been used as approved feed additives to prevent infections and to promote growth in commercially grown poultry and swine [3]. Roxarsone (4-hydroxy-3nitrobenzenearsonate) is the major feed additive used for broiler production [4]. Roxarsone and its metabolites are primarily excreted, adding more than 1100 tons per year to waterways, especially downstream from CAFOs [5,6,7]. Poultry litter (excrement, spilled feed, cage straw) is frequently used as fertilizer [8]. Some states have recently banned or decreased poultry litter use as fertilizer, but enforcement procedures are not clear [9,10]. The areas downstream of poultry CAFOs where the populations’ drinking water is provided by the local groundwater are of particular concern [11]
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